U.S. patent number 5,284,558 [Application Number 07/558,708] was granted by the patent office on 1994-02-08 for electrophoresis-based sequencing of oligosaccharides.
This patent grant is currently assigned to University of Iowa Research Foundation. Invention is credited to Ali Al-Hakim, Kyung-Bok Lee, Robert J. Linhardt, Duraikkannu Loganathan.
United States Patent |
5,284,558 |
Linhardt , et al. |
February 8, 1994 |
Electrophoresis-based sequencing of oligosaccharides
Abstract
The electrophoretic isolation and monosaccharide sequence
determination of a neutral or weakly acidic oligosaccharide species
of interest are disclosed. A labeling compound and a charged group
are coupled to the reducing end of the species of interest, thereby
facilitating electrophoretic separation and detection of the
separated species. The resolved species of interest can then be
recovered from the electrophoretic medium, for example, by
electrophoretic transfer to a charged solid support. Following
isolation, monosaccharide units can be cleaved successively from
the non-reducing end of the species of interest to reveal the
monosaccharide sequence. The identity of each monosaccharide unit
is determined by correlating cleavage data with known
exoglycosidase specificites.
Inventors: |
Linhardt; Robert J. (Iowa City,
IA), Lee; Kyung-Bok (Iowa City, IA), Al-Hakim; Ali
(Iowa City, IA), Loganathan; Duraikkannu (Ann Anbor,
MI) |
Assignee: |
University of Iowa Research
Foundation (Iowa City, IA)
|
Family
ID: |
24230638 |
Appl.
No.: |
07/558,708 |
Filed: |
July 27, 1990 |
Current U.S.
Class: |
204/451; 204/456;
435/18; 435/212; 436/94; 536/127; 536/18.5 |
Current CPC
Class: |
C12Q
1/34 (20130101); G01N 33/52 (20130101); Y10T
436/143333 (20150115); G01N 2333/924 (20130101) |
Current International
Class: |
C12Q
1/34 (20060101); G01N 33/52 (20060101); G01N
027/447 (); G01N 027/26 (); G01N 033/48 (); C12Q
001/34 () |
Field of
Search: |
;435/274,18,212 ;436/94
;204/182.8,182.9,180.1,299R |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Lee et al., Appl. Biochem. and Biotechnol., 23:53-80 (1990). .
Al-Hakim et al., Electrophoresis, 11:23-28 (1990). .
Rice et al., Biochem. J., 244:515-522 (1987). .
Oku et al., Anal. Biochem., 185:331-334 (1990). .
Takara Advertising Literature. .
Welply, TIBTECH, 7:5-10 (1989). .
Koller et al., Chemical Abstracts, 111:649, Abstract 32889y
(1989)..
|
Primary Examiner: Niebling; John
Assistant Examiner: Starsiak, Jr.; John S.
Attorney, Agent or Firm: Hamilton, Brook, Smith &
Reynolds
Claims
We claim:
1. A method for determining the monosaccharide sequence of a
neutral or weakly acidic oligosaccharide species of interest,
comprising:
a) coupling a labeling compound to the reducing end of the species
of interest, the labeling compound bearing a charged group selected
from the group consisting of sulfate, sulfonate, phosphate or
quatenary ammonium groups to produce a charged oligosaccharide
species of interest having size X, wherein X is the number of
monosaccharide units in the species of interest;
b) treating individual samples of the charged oligosaccharide
species of interest with exoglycosidases, each sample of the
derivatized species of interest being treated with a single
exoglycosidase;
c) resolving the reaction products of step b) in an electrophoretic
medium to identify the exoglycosidase which cleaves a single
monosaccharide unit from the non-reducing end of the charged
oligosaccharide species of interest to produce a charged
oligosaccharide species of interest having size X-1;
d) correlating cleavage data with known exoglycosidase
specificities to determine the identity of the monosaccharide
cleaved from the non-reducing terminus;
e)isolating the charged oligosaccharide species of interest having
size X-1; and
f) determining the complete sequence of monosaccharides in the
charged oligosaccharide species of interest by sequentially
repeating steps b)-e) to determine the monosaccharide unit present
at the non-reducing terminus of progressively shorter
oligosaccharide units, each progressively shorter oligosaccharide
unit differing from its parent by a single monosaccharide unit.
2. A method of claim 1 wherein the labeling compound is selected
from the group consisting of the monopotassium salt of
7-amino-1,3-naphthalenedisulfonic acid or the trisodium salt of
1-aminopyrene-3,6,8-trisulfonic acid.
3. A method of claim 2 wherein the electrophoretic medium is an
acrylamide gel.
4. A method of claim 2 wherein the electrophoretic medium is a
capillary electrophoretic medium.
5. A method for determining the monosaccharide sequence of a
neutral or weakly acidic oligosaccharide species of interest, the
species of interest being a component of a glycoconjugate,
comprising:
a) releasing oligosaccharide components from the glycoconjugate by
chemical or enzymatic treatment;
b) coupling a labeling compound to the reducing end of the
oligosaccharide components, the labeling compound bearing a charged
group selected from the group consisting of sulfate, sulfonate,
phosphate or quatenary ammonium groups to produce a charged
oligosaccharide species of interest;
c) separating the oligosaccharide components in an electrophoretic
medium;
d) recovering the charged oligosaccharide species of interest from
the electrophoretic medium;
e) treating individual samples of the charged oligosaccharide
species of interest with exoglycosidases, each sample of the
charged oligosaccharide species of interest being treated with a
single exoglycosidase;
f) separating the reaction products of step e) in an
electrophoretic medium to identify the exoglycosidase which cleaves
a single monosaccharide unit from the non-reducing end of the
charged oligosaccharide species of interest to produce a charged
oligosaccharide species of interest having size X-1;
g) correlating cleavage data with known exoglycosidase
specificities to determine the identity of the monosaccharide
cleaved from the non-reducing terminus;
h) isolating the charged oligosaccharide species of interest having
size X-1; and
i) determining the complete sequence of monosaccharides in the
species of interest by sequentially repeating steps e)-h) to
determine the monosaccharide unit present at the non-reducing
terminus of progressively shorter oligosaccharide units, each
progressively shorter oligosaccharide unit differing from its
parent by a single monosaccharide unit.
6. A method of claim 5 wherein the glycoconjugate is selected from
the group consisting of glycolipids or glycoproteins.
7. A method of claim 6 wherein the enzymatic treatment comprises
contacting the glycoconjugate with an endoglycosidase.
8. A method of claim 7 wherein the endoglycosidase is
N-glycanase.
9. A method of claim 5 wherein the labeling compound is selected
from the group consisting of the monopotassium salt of
7-amino-1,3-naphthalenedisulfonic acid or the trisodium salt of
1-aminopyrene-3,6,8-trisulfonic acid.
10. A method of claim 9 wherein the electrophoretic medium is a
gradient acrylamide gel.
11. A method of claim 9 wherein the electrophoretic medium is a
capillary electrophoretic medium.
12. A method of claim 9 wherein the resolved oligosaccharide
species are recovered from the electrophoretic medium by
electrotransfer to a charged solid support.
13. A method of claim 12 wherein the electrotransfer method is a
semi-dry electrotransfer.
14. A method for determining the monosaccharide sequence of a
neutral or weakly acidic oligosaccharide species of interest, the
oligosaccharide species of interest contained in a mixture of
oligosaccharides, comprising:
a) coupling a labeling compound to the reducing end of the
oligosaccharide components in the mixture, the labeling compound
bearing a charged group selected from the group consisting of
sulfate, sulfonate, phosphate or quatenary ammonium groups to
produce a charged oligosaccharide species of interest;
b) separating the charged oligosaccharide components in an
electrophoretic medium;
c) recovering the charged oligosaccharide species of interest from
the electrophoretic medium;
d) treating individual samples of the charged oligosaccharide
species of interest with exoglycosidases, each sample of the
charged oligosaccharide species of interest being treated with a
single exoglycosidase;
e) separating the reaction products of step d) in an
electrophoretic medium to identify the exoglycosidase which cleaves
a single monosaccharide unit from the non-reducing end of the
charged oligosaccharide species of interest to produce a
derivatized species of interest having size X-1;
f) correlating cleavage data with known exoglycosidase
specificities to determine the identity of the monosaccharide
cleaved from the non-reducing terminus;
g) isolating the charged oligosaccharide species of interest having
size X-1; and
h) determining the complete sequence of monosaccharides in the
charged oligosaccharide species of interest by sequentially
repeating steps d)-g) to determine the monosaccharide unit present
at the non-reducing terminus of progressively shorter
oligosaccharide units, each progressively shorter oligosaccharide
unit differing from its parent by a single monosaccharide unit.
15. A method of claim 14 wherein the labeling compound is selected
from the group consisting of the monopotassium salt of
7-amino-1,3-naphthalenedisulfonic acid or the trisodium salt of
1-aminopyrene-3,6,8-trisulfonic acid.
16. A method of claim 14 wherein the electrophoretic medium is an
acrylamide gel.
17. A method of claim 14 wherein the electrophoretic medium is a
capillary electrophoretic medium.
18. An oligosaccharide having attached, at its reducing end, a
labeling compound bearing a charged group selected from the group
consisting of sulfate, sulfonate, phosphate or quatenary ammonium
groups.
19. An oligosaccharide of claim 18 wherein the labeling compound is
selected from the group consisting of the monopotassium salt of
7-amino-1,3-naphthalenedisulfonic acid or the trisodium salt of
1-aminopyrene-3,6,8-trisulfonic acid.
Description
BACKGROUND OF THE INVENTION
The simple sugars are among the most important small organic
molecules in the cell. The simplest type of sugars, the
monosaccharides, are compounds having the general formula (CH.sub.2
O).sub.n, where n is an integer from three through seven. All
sugars contain hydroxyl groups and either an aldehyde or a ketone
group. Sugar monomers can be combined via a glycosidic bond by the
reaction of a hydroxyl group of one sugar with the aldehyde or
ketone group of a second sugar to form disaccharides,
oligosaccharides and polysaccharides. Because each monosaccharide
has several reactive hydroxyl groups, complex sugars can exhibit
branching rather than a simple linear architecture.
Simple polysaccharides such as glycogen, which exhibits a repeating
structure of glucose monomers, are used principally as energy
stores by the cell. Smaller, but more complex oligosaccharides
function in other important cellular roles. For example, such
oligosaccharides can be linked to proteins or lipids to form
glycoproteins or glycolipids, respectively.
In recent years, a major focus for researchers in the area of
carbohydrate chemistry has been the glycoproteins. The
oligosaccharide side chains of glycoproteins have been implicated
in such cellular processes as protection of peptide chains against
proteolytic attack, facilitation of the secretion of certain
proteins or their mobilization to the cell surface, induction and
maintainance of the protein conformation in a biologically-active
form, clearance of glycoproteins from plasma, direction of the
immune response by acting as immune decays, and function as
antigenic determinants in differentiation and development.
Information about glycoprotein sugar side-chain composition, and
more importantly their sequence is required to fully understand and
establish structure-function relationships. However, glycoproteins
are usually available in only limited quantities (typically 1-100
micrograms glycoprotein, containing 1-10% oligosaccharide) making
it difficult to determine the sequence and anomeric configuration
of glycosidic linkage. Unfortunately, current techniques employed
to determine oligosaccharide sequence require milligram quantities
of the oligosaccharide species. A need exists for a simplified
oligosaccharide sequencing method which is useful to determine the
sequence when only small (e.g., microgram quantities) quantities of
common oligosaccharides are available.
SUMMARY OF THE INVENTION
This invention pertains to methods for the isolation and
purification of a neutral or weakly acidic oligosaccharide species
of interest from a mixture of oligosaccharide components. A
labeling compound and a charged group are coupled to the reducing
end of the oligosaccharide components in the mixture to produce a
derivatized species of interest. The oligosaccharide components are
resolved in a electrophoretic medium and the derivatized species of
interest is then recovered from the electrophoretic medium, for
example, by electrophoretic transfer to a charged solid
support.
In another aspect, the invention pertains to methods for
determining the monosaccharide sequence of a neutral or weakly
acidic oligosaccharide species of interest having size X. As used
herein, the variable X represents the number of monosaccharide
units in the oligosaccharide species of interest. A labeling
compound and a charged group are coupled to the reducing end of the
oligosaccharide species of interest to produce a derivatized
species of interest.
Individual samples of the derivatized species of interest are
treated with exoglycosidases. Each individual sample is treated
with a single different exoglycosidase. The products of this
exoglycosidase treatment are resolved in an electrophoretic medium.
The exoglycosidase which acts on the derivatized species of
interest is identified. In the case of a typical linear
(unbranched) oligosaccharide, only one exoglycosidase will act to
cleave a single monosaccharide unit from the non-reducing end of
the derivatized species of interest to produce a derivatized
species of interest having size X-1. The cleavage data is
correlated with known exoglycosidase specificities to determine the
identity of the monosaccharide cleaved from the non-reducing
terminus.
The derivatized species of interest having size X-1 is isolated and
the complete sequence of monosaccharides in the oligosaccharide of
interest is determinded by sequentially repeating the
exoglycosidase treatment and electrophoretic analysis steps
specified above to determine the monosaccharide unit present at the
non-reducing terminus of progressively shorter oligosaccharide
units, each progressively shorter oligosaccharide unit differing
from its parent by a single monosaccharide unit.
The methods described herein facilitate the separation and
monosaccharide sequence determination of microgram quantities of an
oligosaccharide of interest, whereas the prior art methods required
milligram quantities. Furthermore, the separation methods described
herein enable the separation of complex oligolosaccharide mixtures
heretofore unresolveable. In addition, the methods described herein
do not require special detection systems, but rather employ basic
apparatus used routinely in biochemical research.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a diagram representing an electrophoretic display of the
reaction products following exoglycosidase treatment of a labeled
nonasaccharide species.
FIG. 2 is a diagram representing a method for labeling an
oligosaccharide or polysaccharide with a charged fluorescent
tag.
FIG. 3 is a diagram representing a strong anion exchange HPLC
analysis of sugar conjugates.
FIG. 4 is a diagram representing sequence analysis of a sugar-AGA
conjugate by gradient polyacrylamide gel electrophoresis.
FIG. 5 is a diagram showing a scheme for sequencing a typical
bianternnary oligosaccharide species that has been labeled and
charged.
DETAILED DESCRIPTION OF THE INVENTION
This invention is based on the discovery that neutral or weakly
acidic oligosaccharide species can be derivatized to facilitate
electrophoretic separation. In one aspect, the methods of this
invention pertain to the isolation of an oligosaccharide of
interest from a mixture of oligosaccharides. In another aspect, the
methods of this invention pertain to the determination of the
sequence of monosaccharides in an oligosaccharide of interest.
In another aspect, this invention pertains to methods for
determining the monosaccharide sequence of a neutral or weakly
acidic oligosaccharide species of interest present as a component
of a glycoconjugate. The oligosaccharide components are released
from the glycoconjugate by chemical or enzymatic treatment. A
labeling compound and a charged group are coupled to the reducing
end of the oligosaccharide component to produce a derivatized
species of interest. The derivatized species of interest is
isolated from the other oligosaccharide components in an
electrophoretic medium and its sequence is determined as described
above.
The term oligosaccharide, as used herein, is defined as a molecule
composed of more than a single saccharide unit where these units
are connected through glycosidic linkages. As used within this
application, the term oligosaccharide would also include high
molecular weight oligomers known as polysaccharides. The only
requirement is that the oligosaccharide (or polysaccharide) must
either have a reducing end or it must be possible to generate a
reducing end by chemical or enzymatic means.
One of the problems associated with the sequencing of
oligosaccharides is that typically oligosaccharide preparations
contain a mixture of oligosaccharide species of different sizes
(polydispersity). In addition, chains having the same monomer
number may have different primary structure of sequences
(heterogeneity). The fractionation of such mixed populations of
oligosaccharide species by gel electrophoresis has been described
for acidic oligosaccharide species (i.e. oligosaccharides having a
pKa of about 2.5 or less). However, most oligosaccharides are
neutral or weakly acidic and do not carry a charge sufficient to
facilitate electrophoretic separation.
The methods of this invention, in one aspect, facilitate the
isolation of a neutral or weakly acidic oligosaccharide species
(i.e. oligosaccharides having a pKa of about 2.5 or greater) by
electrophoresis. This is accomplished by first coupling a charged
group and a labeling compound to the reducing ends of the
oligosaccharide species in the mixture. This step accomplishes two
objectives. First, the labeling group attached to the reducing end
of the oligosaccharide provides a marker at a specific location in
the molecule which can be detected, for example, in an
electrophoretic medium. Secondly, the electrical charge of the
labeling compound is responsible for the migration of the
oligosaccharide species when placed under the influence of an
electrical field.
The reducing end can be labeled with any labeling compound
including, for example, fluorescent, radioactive or UV-active
compounds. The charged group can be added in a single step reaction
with the labeling compound as described below, or the labeling
compound and the charged group can be added to the reducing end in
separate steps. Preferred charged groups include sulfate, phosphate
or quatenary ammonium groups.
In a preferred embodiment, the reducing ends of the oligosaccharide
species are derivatized by reaction with fluorescent, negatively
charged molecules. For example, two such compounds are
7-amino-1,3-naphthalenedisulfonic acid, monopotassium salt and
1-aminopyrene-3,6,8,-trisulfonic acid, trisodium salt. These two
compounds derive their negative charge from the sulfate groups,
however, other charged groups such as phosphate, quatenary
ammonium, etc., can be used. The details of such a derivatization
reaction are described in the Exemplification below.
The oligosaccharide mixture bearing the charged labeling group is
then resolved into its constituent species by electrophoresis. In a
preferred embodiment, the electrophoretic medium is a
polyacrylamide gel. The polyacrylamide gel can be of uniform pore
size, or a gradient gel can be used. A particularly useful gradient
range is from 12% cross-linking at the top, to 22% cross-linking at
the bottom, and includes a conventional stacking gel.
An alternative method relies on capillary electrophoresis to
resolve an oligosaccharide mixture bearing the charged labeling
group into its constituent species. Electrophoresis is done in a
narrow bore capillary tube. This tube can be empty as in capillary
zone electrophoresis (CZE). In CZE the interaction of the charged
oligosaccharide with the internal wall of the capillary under the
influence of an electric field results in a separation based
primarily on molecular charge. The capillary can be filled, either
with a polyacrylamide gel (capillary gel electrophoresis) or with a
viscose solution of polyethylene glycol or dextran (capillary
dynamic sieving electrophoresis) and a separation based primarily
on oligosaccharide size results.
Following electrophoresis, the resolved oligosaccharide species are
recovered from the electrophoretic medium. A preferred method for
recovering the resolved oligosaccharide species from a gel is by
electrotransfer to a charged solid support. Particularly preferred
is the semi-dry electrotransfer described in detail in the
Exemplification. The electrotransfer of charged molecules, other
than oligosaccharides, from a gel to a charged solid support is
well known in the art. Examples of appropriate charged solid
support materials include nylon membranes, nitrocellulose, etc. The
support bound oligosaccharides are released from the support by
incubation in an appropriate buffer, desalted, for example, by
dialysis and concentrated, for example, by lyophilization. One
skilled in the art would know or could devise numerous methods for
recovering the resolved oligosaccharide species from the
preparative acrylamide gel.
In the case of capillary electrophoresis, resolved oligosaccharide
species can be recovered by simply repetitively running them out of
the capillary tube into a fraction collector.
The recovered oligosaccharide, having a charged label can be used
in several ways. First, the label can be removed by chemical or
enzymatic methods. For example, the labeled bianternnary
oligosaccharide, shown in FIG. 5, could be treated with hydrazine
or with endoglycosidase F(EC3.2.1.96, Genzyme). This affords a pure
oligosaccharide for various applications. Second, the recovered
labeled oligosaccharide can be used as a substrate for
exoglycosidases and would be useful in assessing purity of
exoglycosidase preparations. Finally, the labeled oligosaccharide
can be sequenced.
In another aspect of the present invention, a method is described
for determining the monosaccharide sequence of a neutral or weakly
acidic oligosaccharide species of interest having a size X. The
variable term X is used herein to denote the number of
monosaccharide units in the oligosaccharide of interest. The
oligosaccharide species of interest, is derivatized as described
above, to couple a labeling compound and a charged group to the
reducing end of the oligosaccharide.
To determine the monosaccharide sequence, samples or aliquots of
the derivatized oligosaccharide species of interest are treated
with exoglycosidases, each sample or aliquot being treated with a
single exoglycosidase. Enzymatic hydrolysis can be carried out on
very small quantities of the recovered oligosaccharide species.
Exoglycosidases are hydrolases which cleave monosaccharide units
from the non-reducing terminus of oligosaccharides. Since
exoglycosidases cleave only monosaccharide residues that are
located at the non-reducing terminus, they are useful tools for
sequencing the oligosaccharide chain. In addition, information
about the anomeric configuration of glycosidic linkages can be
obtained by using the known specificity of glycosidases. A partial
list of exoglycosidases known to be useful are presented in Table
1.
TABLE 1
__________________________________________________________________________
EXOGLYCOSIDASES Enzyme Source Specificity References
__________________________________________________________________________
.alpha.-L-fucosidase Charonia very broad, (1, 2) (EC 3.2.1.51)
lampas all fucosyl linkages .alpha.-D-galactosidase Coffee bean
very broad aglycone (3) (EC 3.2.1.22) .alpha.-mannosidase Jack bean
Man-.alpha.-(1.fwdarw.2)-Man, Man-.alpha. (4) (EC 3.2.1.24)
(1.fwdarw.6)-Man with 100%, and Man-.alpha.-(1.fwdarw.3)-Man with
7% reaction rate .alpha.-glucosidase Yeast .alpha.-(1.fwdarw.2),
.alpha.-(1.fwdarw.3), (5) (EC 3.2.1.20) .alpha.-(1.fwdarw.4)
glycopyranosides, whereas .alpha.-(1.fwdarw.6) bonds are only
attacked slowly Neuraminidase Clostridium
NeuAc-.alpha.-(2.fwdarw.3)-Gal (6) (EC 3.2.1.18) perfringens
NeuAc-.alpha.-(2.fwdarw.6)-Gal, and
NeuAc-.alpha.-(2.fwdarw.6)-GalcNAc .beta.-glucuronidase E. coli K
12 .beta.-D-glucuronides (7) (EC 3.2.1.31) .beta.-glucosidase
Amyodalae .beta.-glucosides of phenols, (8) (EC 3.2.1.21) dulces
salicyl alcohol, vanilin, 2-cresol and 4-cresol
.beta.-galactosidase Bovine testes Gal-.beta.-(1.fwdarw.3)-GlcNAc
(9, 10) (EC 3.2.1.23) Gal-.beta.-(1.fwdarw.4)-GlcNAc
Gal-.beta.-(1.fwdarw.4)-GalNAc Streptococcus
Gal-.beta.-(1.fwdarw.4)-GlcNAc pneumoniae No cleavage of
Gal-.beta.-(1.fwdarw.3)-GlcNAc or Gal-.beta. -(1.fwdarw.6)-GlcNAc
.beta.-N-acetyl-D- Beef kidney N-acetyl-.beta.-D-glu (11)
glucosaminidase cosaminides, N-acetyl- (EC 3.2.1.30)
.beta.-D-galactosamindes .beta.-N-acetyl Aspergillus GalNAc-ser
link (12) galactosaminidase niger (EC 3.2.1.4)
__________________________________________________________________________
(1) Iijima, Y. and F. Egami, J. Biochem. (Tokyo) 70:75 (1971). (2)
Nishigaki, M. et al., J. Biochem. (Tokyo) 75:509 (1974). (3)
Courtois, J. E. and F. Pertek, Methods Enzymol 8:565 (1966). (4)
Yamashita, K. et al., J Biol. Chem. 255:5635 (1975). (5) Halvorson,
H., Methods Enzymol. 8:559 (1966). (6) Cassidy, J. T. et al., J
Biol. Chem. 240:3501 (1965). (7) Bergmeyer, H. U. et al., Methods
of Enzymatic Analysis. Bergmeyer, H.U., ed., VCH, New York, pp.
246-256. (8) Conchie, J. et al., Biochem. J. 103:609 (1967). (9)
Uchida, Y. et al., J. Biochem (Tokyo) 80:1573 (1979). (10) Paulson,
J. C. et al., J. Biol. Chem. 253:5617 (1978). (11) Glasgow, L.R. et
al., J. Biol. Chem. 252:8615 (1977). (12) McDonald, M. J. and O. P.
Bahl, Methods Enzymol 28:734 (1972).
The products of the individual exoglycosidase reaction mixtures are
then separated by electrophoresis. As discussed above, in
connection with the purification of individual species, preferred
embodiments include polyacrylamide gradient gel, electrophoresis
and capillary electrophoresis. Since the label is located at the
reducing end of the oligosaccharide chain, the digestion products
are a monosaccharide and a smaller labeled oligosaccharide having
size X-1. Because each exoglycosidase specifically recognizes and
cleaves only one type of monosaccharide from the non-reducing end
of the oligosaccharide chain, the identity of the terminal
monosaccharide is revealed by determining which of the tested
enzymes resulted in a cleavage of the oligosaccharide (as evidenced
by the altered mobility of the labeled fragment in the
electrophoretic medium).
More specifically, for example, FIG. 1 represents the
electrophoretic separation of the reaction products from a battery
digest of an nonasaccharide (as shown in step 2, FIG. 5) containing
the monosaccharides galactose at its two non-reducing ends. Lane 5
represents the reaction mixture containing .beta.-galactosidase
which specifically releases .beta.-D-galactose. The released
monomers would not be detectable in the gel because they are
unlabeled. If it becomes desirable to detect released
monosaccharide, labeling with the same tag (or a different tag)
containing a charged group, can be performed after its enzymatic
release. The labeled oligosaccharide represented in lanes 1-4 is
not cleaved by the other exoglycosidases and, therefore, its
electrophoretic mobility is unchanged. Lane 6 represents a sample
of the labeled oligosaccharide which was not treated with an
exoglycosidase and which was run on the gel as a mobility
standard.
The labeled oligosaccharide contained within the lane of the gel
corresponding to the reaction mixture containing the enzyme which
recognized and cleaved the terminal monosaccharides (lane 5 in this
case) is then recovered from the gel, for example, using semi-dry
electrotransfer as described herein. The battery digestion
procedure and electrophoretic display is then repeated to determine
each of monosaccharides in the chain thereby revealing the sequence
of the oligosaccharide species.
The structure of many oligosaccharide species can be deduced with
greater certainty by using enzymes having greater specificity. For
example, chains with 1.fwdarw.3 linked galactose at their
non-reducing end can be distinguished from ones with a 1.fwdarw.6
linked galactose by using galactosidase (EC3.2.1.23, bovine testes)
in place of galactosidase (EC3.2.1.23, E. coli). Alternatively,
endoglycosidases, such as .beta.-endogalactosidase (E. freundii,
EC3.2.1.103) can be used in sequencing. Another complicating
structural feature is one in which a labeled oligosaccharide
contains two (or more) identical sugars at its non-reducing end and
where these sugars are linked in the same configuration. As soon as
the exoglycosidase removes the non-reducing end sugar, it exposes a
new, identical non-reducing end on which it can act. In such cases,
sequencing is accomplished by incompletely treating the labeled
oligosaccharide with exoglycosidase. Analysis then shows multiple
bands corresponding to unreacted starting material, oligosaccharde
of size X-1, and oligosaccharide of X-2. The sequencing of the
labeled oligosaccharide, terminating in two .beta. (1.fwdarw.4)
linked galactose residues, illustrates this approach.
Sequencing branched oligosaccharides follow the same general scheme
as used for linear oligosaccharides. In a branched oligosaccharide
(in which each branch terminates with a different sugar) when
treated with the set of exoglycosidases described in the previous
example (FIG. 1), will show two lanes to have a shifted band. Both
shifted bands will then be recovered, by electrotransfer, for
example, and sequenced individually. This will result in the
sequence of each branch.
The discussion above relating to sequencing has been limited to the
analysis of a sample containing a single oligosaccharide species.
The method of this invention is also useful for determining the
monosaccharide sequence of an oligosaccharide sequence of interest
contained within a mixture of oligosaccharides. This can be
accomplished by essentially by combining the isolation and
sequencing methods described above. That is, the oligosaccharide
species present in the mixture are derivatized by the addition of a
labeling compound and a charged group at the reducing terminus.
These derivatized species are next separated in an electrophoretic
medium. The derivatized oligosaccharide of interest is recovered
from the medium and subjected to exoglycosidase treatment as
described above to determine the monomer sequence.
The methods of this invention are also useful for the determination
of the sequence of an oligosaccharide species present as a
component of a glycoconjugate. Two very important classes of
glycoconjugate are the glycolipids and glycoproteins. The core
element of a glycoprotein is a protein which has been modified by
cellular enzymes by the covalent addition of one or more
oligosaccharide moieties to the side chains of particular amino
acid residues by a process known as glycosylation. The most common
oligosaccharide linkage is to the side chain of the amino acid
asparagine; such linkages are referred to as N-linked
oligosaccharides. Oligosaccharides are also known to be linked to
the OH group on the side chain of serine, threonine, or
hydroxylysine residues; these are referred to as O-linked
oligosaccharides.
To determine the sequence of an oligosaccharide species which is a
component of a glycoprotein, the oligosaccharide species must first
be released from the core element of the glycoprotein. Some
glycoproteins contain multiple oligosaccharide components.
Glycoproteins can be purified using techniques well known to those
skilled in the art. In order to release the oligosaccharide species
from the protein core, the glycoprotein is treated with a chemical
or enzyme capable of breaking the glycosidic bond which links the
oligosaccharide species to the protein core.
Hydrazine treatment can be used, for example, to release
oligosaccharides from glycoproteins. Hydrazine cleaves the
N-glycosidic linkages of N-glycosylpeptides and N-glycosylproteins
and liberates the N-deacetylated glycans as their hydrazones.
Treatment with (1:1) mixtures of trifluoroacetic anhydride and
trifluroacetic acid at 100.degree. C. for 48 h also results in
cleavage of the glycosylamine protein-carbohydrate linkage.
Trifluoroacetolysis cleaves peptide bonds by transamidation and
replaces the N-acetyl substituents of amino sugars by
N-trifluoroacetyl groups.
O-glycosidic linkages between carbohydrate chain and the
beta-hydroxyamino acids serine and threonine are easily cleaved
using dilute alkali solution (o.05-0.1M NaOH or KOH) under mild
conditions (4.degree.-45.degree. C. for 0.5-6 d) by a
beta-elimination mechanism.
A preferred method for removing oligosaccharide species from a
glyconjugate is by treatment with an endoglycosidase. The
endoglycosidase specifically cleaves the glycosidic bond which
joins the oligosaccharide to the core element. A preferred
endoglycosidase is the enzyme glycopeptidase F(EC3.2.2.18)
N-glycanase.TM., (Genzyme) which removes N-linked oligosaccharides
or endo-.alpha.-N-acetylgalactosaminidase (O-glycanase.TM.,
Genzyme).
Similar methods can be used to remove oligosaccharides from
glycolipids. Chemical methods include treatment of glycolipid with
trifluoroacetic acid/trifluoro acetic anhydride, osmium
tetroxide/periodic acid, or ozone/sodium hydroxide or sodium
carbonate. Enzymatic methods include the use of ceramide glycanase
(endoglycoceramidase, Boerhinger Mannheim).
The invention is illustrated further by the following
Exemplification.
EXAMPLES
Example 1
Materials and Methods
Chemicals
N-acetyl-D-glucosamine (2-acetamido-2-deoxy-D-glucose),
N-acetyl-D-galactosamine (2-actamido-2-deoxy-D-galactose), D
(+)-mannose, D (+)-glucose, D (+)-galactose, L (+)-arabinose,
N-acetyl-lactosamine
(2-acetamido-2-deoxy-4-O-.beta.-D-galactopyranosyl-D-glucopyranose;
.beta.-D-galactopyranosyl-[1.fwdarw.4]-N-acetyl-D-glucosamine),
lacto-N-biose(2-acetamido-2-deoxy-3-O-.beta.-D-galactopyranosyl-.beta.-D-g
lucopyranose;
.beta.-D-galactopyranose-[1.fwdarw.3]-N-acetyl-D-glucosamine),
maltose (4-O-.alpha.-D-glucopyranosyl-D-glucose),
2-acetamido-2-deoxy-4-O-([4-O-.beta.-D-galactopyranosyl]-.beta.-D-galactop
yranosyl-D-glucopyranose; .beta.-D-galactopyran
ose-[1.fwdarw.4]-.beta.-D-galactopyranose-[1.fwdarw.4]-N-acetyl-D-glucosam
ine, maltoheptose, maltooligosaccharide mixtures
({-.beta.-D-glucopyranose-[1.fwdarw.4]}.sub.4-10), sodium
cyanoborohydride, .beta.-galactosidase (from jack beans,
Escherichia coli and bovine testes) were obtained from Sigma
Chemical Co., St. Louis, MO, USA. Monopotassium
7-amino-1,3-naphthalenedisulfonic acid (Amido-G-acid; AGA), and
.sup.2 H.sub.2 O (99.996%) were purchased from Aldrich, Milwaukee,
WI, USA. Trisodium 1-aminopyrene-3,6,8-trisulfonic acid (APTS) was
purchased from Lamda Probes & Diagnostics, Grottenhof, Austria.
Spectrapore dialysis tubing (Mr cut-off 100 and 500) was purchased
from Spectrum Medical, Los Angeles, CA, USA. Bio-P2-gel was from
Biorad, Richmond, CA, USA. Acrylamide (ultrapure), Tris, alcian
blue dye, bromophenol blue dye and ammonium persulfate were
obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN,
USA. Glycine hydrochloride, disodium EDTA, boric acid, sucrose,
N,N-methylene bisacrylamide and N,N,N,N-tetramethylenediamine
(TEMED) were from Fisher Chemical Company, Fair Lawn, NJ, USA.
Biotrace RP nylon and nitrocellulose membranes were obtained from
Gelman Science Inc., Ann Arbor, MI, USA., 3 mm paper, from Whatman,
Hillsboro, OR, USA. Bio-P2 gel was purchased from Biorad, Richmond,
CA, USA. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was
obtained from Merck Sharp & Dohme, Kirkland, Quebec, Canada.
All other chemicals were reagent-grade.
Equipment
Strong-anion-exchange high performance liquid chromatography
(SAX-HPLC) was performed using two Shimadzu Bio Liquid
Chromatograph LC-7A pumps (Kyoto, Japan) with gradient control by
digital to analog interface using an Apple IIe microcomputer
running Chromatochart software from Interactive Microware Inc.,
State College, PA, USA. The system was equipped with a fixed-volume
loop Rheodyne (Cotai, CA) #7125 injector and 2141 variable
wavelength detector from Pharmacia LKB Biotechnology, Inc.,
Piscataway, NJ, USA. The data was processed using a Shimadzu
Chromatopac C-R3A integrating recorder. SAX-HPLC was performed on a
Spherisorb (5 .mu.m particle size) column of dimensions 4.6
mm.times.25 cm, with a 4.6 mm.times.5 cm guard column from Phase
Separations, Norwalk, CT, USA. A 32 cm.times.16 cm vertical slab
gel unit (SE 620), 250 ml SG500 linear gradient maker apparatus and
the TE70 semi-dry electrophoretic transfer unit were obtained from
Hoefer Scientific Instruments, San Francisco, CA, USA. An
electrophoresis power unit model 1420B and trans-blot
electro-transfer system were purchased from Biorad, Richmond, CA,
USA. Sugar-florescent conjugates were visualized in the ultraviolet
light chamber from Ultra-violet Products, Inc., San Gabriel, CA,
USA. Freeze-drying was done on a Virtis Freezemobile 6
freeze-drier.
Methods
Preparation of Fluorescence Labeled Standard Sugars by Reductive
Amination Method
AGA was used after recrystallization from deionized water (The
Merck Index, 11th Edition, Merck & Co., Inc., Rahway, NJ, p.
66, (1989)). A standard sugar (3.5 .mu.mol) was dissolved in 720
.mu.l AGA or APTS solution (50 wt %, pH 6.2). After heating at
80.degree. C. for 1 h, 36 .mu.l of reducing agent was added. The
reducing agent was prepared by mixing 10 mg of sodium
cyanoborohydride, 20 .mu.l AGA solution and 30 .mu.l of water. The
mixture was heated and agitated at 100 rpm for 24 h at 65.degree.
C. in an incubator shaker. After the reaction was complete, the
products were dialyzed overnight at 40.degree. C. against double
distilled deionized water in either 100 (for monosaccharides and
disaccharides) and 500 (for trisaccharides) Mr cut-off controlled
pore dialysis bags. The samples were freeze-dried and reconstituted
in 100 .mu.l of distilled water before loading on the preparative
gel.
Preparation and Electrophoresis of Gradient Polyacrylamide Gels
Gradient polyacrylamide resolving gel was prepared from two
different resolving gel concentrations. The back chamber contained
11.5% (W/V) N,N-bisacrylamide, 0.5% (W/V) N,N-bisacrylamide and 1%
(W/V) sucrose in resolving buffer (lower buffer chamber) made from
0.1M boric acid, 0.1M Tris and 0.01M disodium EDTA, pH 8.3 (Rice,
K.G. et al., Biochem. J. 244:515-522 (1987)). The mixing chamber
contained 20% (W/V) of N,N-acrylamide, 2% (W/V) N,N-bisacrylamide
and 15% (W/V) of sucrose, pH 8.3, in resolving buffer. Gels were
poured vertically between two glass plates (16 cm.times.32 cm)
separated by 1.5 mm spacers. Gradients were poured by adding 35 ml
of 12% solution (degassed) to the reservoir (back chamber) and 35
ml of 22% solution (degassed) to the mixing chamber. Ammonium
persulfate, 400 .mu.l of 10% solution, was added to the reservoir
and 200 .mu.l to the mixing chamber followed by addition of 30
.mu.l of TEMED to both the reservoir and mixing chamber. The
solutions were mixed thoroughly. The acrylamide solution in the
mixing chamber was continuously mixed using a magnetic stirrer. The
valve between the reservoir and the mixing chamber was opened.
Polyacrylamide solution from the mixing chamber passed by gravity
into two channels leading to the top of the glass plates forming a
linear gradient from bottom to top. The unpolymerized solution was
overlaid with water. Polymerization occurred from top to bottom.
After polymerization had completed, the water layer was removed.
Ten ml of stacking gel made from 4.75% (W/V) N,N-acrylamide and
0.25% (W/V) N,N-bisacrylamide in resolving buffer but adjusted to
pH 6.3 with hydrochloric acid was mixed. Ammonium persulfate, 135
.mu.l of 10%, and 10 .mu.l of TEMED was added to the top of the
resolving gel. A desired comb (well former) was inserted. After
polymerization, the comb was removed and the walls were washed with
water and upper buffer chamber was filled with a buffer made from
1.25M glycine and 0.2M Tris pH 8.3. Samples combined with an equal
volume of 50% sucrose solution containing trace quantities of
phenol red and bromophenol blue were loaded carefully to the bottom
corner of each well. Electrophoresis was performed for 18 hours at
400 volts with cooling using circulating tap water. The gel was
removed from the glass plate and, if necessary, stained with alcian
blue 0.5% (W/V) in 2% V/V aqueous acetic acid for 30 minutes.
Destaining was carried out using several 200 ml volumes of
distilled water.
Semi-Dry Electro-Transfer
The resolving gel was visualized using a ultraviolet light and the
desired fluorescent band was located. The band was removed from the
gel by carefully cutting the gel. This slice was then soaked in
transfer buffer consisting of Tris base (5.82 g), glycine
hydrochloride (4.35 g) and methanol (200 ml) made up in 1 l with
double distilled deionized water. Several layers of blotting paper
and positively charged transfer nylon membranes were cut to the
same size as the gel and soaked in transfer buffer. Two pieces of
transfer buffer-saturated blotting papers (3 mm) were placed on the
top of the Mylar Mask, centering them over the opening. Multiple
layers of saturated nylon membranes (depending on the quantity of
sample on the gel) were placed on the top of the blotting papers.
The soaked gel was placed directly on the nylon membranes followed
by 3 layers of blotting papers, thus constructing a transfer
sandwich. The cover of the semi-dry transfer unit was placed over
the transfer sandwich and electro-transfer was performed at 7-10 V
for 1 hour. Completion of the transfer process was ensured by
examining the gel under unltraviolet light and making sure that no
material was left behind. The nylon membranes were removed and the
sample was recovered as described below.
Elution and Recovery of Membrane-Bound Sugar-Fluorescent
Conjugates
Bands on the nylon membrane were located and visualized using
ultraviolet light at 366 nm. Nylon membranes were cut into small
pieces and immersed in test tubes containing 3 ml of 2.0M sodium
chloride and placed on a shaker for several hours at room
temperature. The salt solution containing recovered materials were
dialyzed against double distilled deionized water. In the case of
the trisaccharide derivative, desalting was performed on a 2.5
cm.times.75 cm Bio-P2 gel low pressure column. The salt-free sample
solution was concentrated by freeze drying.
Spectroscopic Methods
The ultraviolet spectra of the AGA and purified sugar-AGA
conjugates were obtained with a Shimadzu UV-160 spectrophotometer
(Kyoto, Japan). The samples were prepared in double distilled
deionized water by serial dilution. All spectroscopic measurements
were performed in 1.0 ml quartz cuvette at room temperature.
Fluorescence characteristics of AGA and purified sugar-AGA
derivatives that include excitation and emission spectra were
recorded in a standard 3.0 ml quartz cuvette at room temperature by
using Shimadzu RF-540 spectrofluorophotometer interfaced with a
Shimadzu Data Recorder DR-3 (Kyoto, Japan). The excitation and
emission slits were 10 nm. The samples were prepared by serial
dilution in double distilled deionized water. One and
two-dimensional NMR spectroscopy was performed on a Bruker WM-360
or MSL-300 spectrometer operating under ASPECT 2000 or 3000
control. Samples were prepared in .sup.2 H.sub.2 O (>99.996%)
containing DSS as the internal standard at room temperature.
Two-dimensional COSY-45 experiment was run using standard Bruker
software. Mass spectrometry was performed on a VG ZAB-HF
spectrometer in the fast atom bombardment (FAB) ionization mode.
Negative-ion FAB spectra were obtained using triethanolamine as the
matrix (Mallis, L.M. et al., Anal. Chem. 61:1453-1458 (1989)).
SAX-HPLC Analysis
SAX-HPLC was performed to monitor the derivatization reaction and
to determine the purity of sugar-fluorescent conjugates obtained
using preparative gradient gel electrophoresis. The column was
eluted with a linear gradient [concentration (y, in M) at any time
(X, in s) +0.0002 X=0.2] of sodium chloride at pH 3.5 and a flow
rate of 1.5 ml/min (Linhardt, R.J. et al., Biochem. J. 254:781-787
(1988)). The elution profile was monitored by absorbance at 247 nm
at 0.02 absorbance units full scale (AUFS).
Enzymatic Digestions
Approximately 5 .mu.g of
.beta.-D-galactopyranose-[1.fwdarw.4]-.beta.-D-galactopyranose-[1.fwdarw.4
]-N-acetyl-D-glucosamine-AGA derivative was treated overnight with
0.1 unit of jack bean .beta.-galactosidase at 25.degree. C. in a
100 .mu.l of 0.2M citrate buffer, pH 3.5. The same amount of
.beta.-D-galactopyranose-[1.fwdarw.4]-.beta.-D-galactopyranose-[1.fwdarw.4
]-N-acetyl-D-glucosa mine-AGA derivative was also treated overnight
with 100 units of Escherichia coli .beta.-galactosidase at
37.degree. C. in a 100 .mu.l of 0.1M phosphate buffer, pH 7.3. Both
enzymatic reactions were performed with agitation in an incubator
shaker at 100 rpm.
Results
The molar ratio of sugar, AGA and sodium cyanoborohydride was
optimized by using N-acetylglucosamine and N-acetylactosamine as
reference sugars. The reaction was monitored by running each sample
on the gradient PAGE and visualizing the products in the
ultraviolet light chamber at 366 nm. To drive the reaction to
completion, a large excess of ACA and longer reaction times were
applied to the larger oligosaccharides. Six monosaccharides
(N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D (+)-mannose, D
(+)-glucose, D (+)-galactose, and L (+)-arabinose), three
disaccharides (N-acetylactosamine, lacto-N-biose, and maltose), one
trisaccharide
(2-acetamido-2-deoxy-4-O-{[4-O-.beta.-D-galactopyranosyl]-.beta.-D-galacto
pyranosyl)-D-glucopyranose;
.beta.-D-galactopyranose-[1.fwdarw.4]-[1.fwdarw.4]-N-acetyl-D-glucosamine)
, maltoheptose, and maltooligosaccharide mixtures were successfully
labeled with the AGA reagents.
Gradient polyacrylamide gel electrophoresis has been used
extensively in the present work due to its effectiveness and
simplicity in resolving, separating and identifying the desired
products. The PAGE system acts as a purifying tool for the crude
reaction mixture as well as a visualizing system for the resolved
fluorescent bands. Previous workers (Linhardt, R.J. et al.,
Biochem. J. 254:781-787 (1988)) have used multiple separation steps
including extraction, high pressure liquid chromatography and thin
layer chromatography to obtain the final products. The method
described here replaces these time consuming, labor intensive
procedures with a single electrophoresis step. Crude products are
loaded directly on gradient gel and fractionated by
electrophoresis. The sugar-fluorescent conjugates appear as well
resolved bands under ultraviolet light. The desired product can be
isolated by electro-transfer from the gel onto a positively charged
nylon membrane. Analysis by SAX-HPLC shows the difference in purity
between the crude and the isolated products. Electrophoresis is
usually carried out for 18 hours and the gel can be visualized
instantly, without any further development, under ultraviolet
light. Electro-transfer using the semi-dry procedure is a 15-minute
technique capable of quantitatively recovering purified
sugar-fluorescent conjugate. Preparative gradient PAGE on 1.5 mm
gels can be used to fractionate 100 mg of sample while 3 mm thick
gels permit the loading of up to 1 g of sugar-fluorescent
conjugate. Analytical gradient PAGE is also valuable for analyzing
nanogram quantities of sugar-fluorescent conjugate.
The structures of sugar-AGA conjugates were established using
spectroscopic and enzymatic methods. Ultraviolet absorption spectra
of the AGA and the sugar-AGA conjugates were compared. Ultraviolet
spectrum of AGA exhibits maximum at 247 nm
(.epsilon.=0.31.times.10.sup.2 M.sup.-1 cm.sup.-1). The
trisaccharide-AGA conjugate, the disaccharide-AGA conjugate
(N-acetyllactosamine-AGA) and the monosaccharide-AGA conjugate
(N-acetylglucosamine-AGA) all shows a 256 nm maxima.
The fluorescence spectra of sugar-AGA conjugates were also
obtained. AGA itself shows the emission maxima at 447 nm and a
excitation maxima at 343 nm. Sugar-AGA conjugates (mono-, di-, and
trisaccharides), show an emission maxima is at 452 nm and an
excitation maxima at 365 nm. Excitation and emission spectra of all
of the sugar-AGA conjugates are very similar. Sugar-AGA conjugates
can be detected using a fluorimeter at femtomolar concentrations.
But, in the ultraviolet light chamber at 366 nm picomole amounts of
sugar-AGA conjugate are easily detected by the human eye.
Purity of the sugar-AGA conjugates obtained using preparative
gradient PAGE was evaluated by SAX-HPLC. AGA and sugar-AGA
conjugates could be separated by SAX-HPLC. AGA-N-acetylglucosamine,
AGA-N-acetyllactosamine, and
.beta.-D-galactopyranose-[1.fwdarw.4]-.beta.-D-galactopyranose-[1.fwdarw.4
]-N-acetyl-D-glucosamine, purified by gradient PAGE, were separated
under sodium chloride linear gradient conditions. SAX-HPLC of crude
reaction mixtures (without any purification) resulted in two major
peaks corresponding to AGA and sugar-AGA conjugate.
The negative-ion FAB-MS spectrum of the trisaccharide-AGA
derivative showed a molecular ion at m/z 853 [M-Na].sup.-
consistent with its molecular weight of 876. The ion at m/z 912
corresponds to addition of one molecule of NaCl to the molecular
ion at m/z 853. The fragment ion at m/z 692 is the result of the
loss of one galactose residue at the non-reducing end. The ion
corresponding to the loss of second galactose residue was not
observed in the spectrum. A detailed NMR study was also undertaken
to unambiguously characterize the sugar-AGA conjugate, particularly
to determine the structure of the linking chain. Earlier studies on
the reductive amination reported that monosaccharide or
oligosaccharides containing N-acetylglucosamine at the reducing end
give rise to low yields (Rosenfelder, G. et al., Anal. Biochem.
147:156-165 (1985)). The exact structure of linking sugar chain in
the products had not been unambiguously established.
N-acetylglucosamine-AGA and the trisaccharide-AGA conjugate,
purified by preparative PAGE method, were examined by .sup.1 H NMR
spectroscopy. In both cases, the absence of anomeric proton signal
of N-acetylglucosamine residue and a slight upfield shift of H-6
and H-8 protons (compared to AGA) support their structure.
Two-dimensional COSY spectrum of N-acetylglucosamine-AGA derivative
fully established its structure.
Sugar-AGA conjugates can be used for sequencing oligosaccharides by
following exoglycosidase treatment on the gel electrophoresis. For
this sequencing strategy to be successful, the sugar-AGA conjugates
must retain their sensitivity to exoglycosidases. To test this,
trisaccharide-AGA conjugate was treated with three different
.beta.-galactosidases (jack bean, E. coli and bovine testes).
Treatment with jack bean and E. coli .beta.-galactosidase resulted
in the disappearance of the trisaccbaride-AGA conjugate band and
the appearance of a lower molecular weight band corresponding to
the monosaccharide-AGA conjugate. This indicated that the
carbohydrate portion of trisaccharide-AGA derivative retains its
sensitivity to .beta.-galactosidase (an exoglycosidase). Therefore,
sugar-AGA conjugates can be used for sequencing analysis by
exoglycosidase digestion. Because .beta.-galactosidase acts at a
site near to the derivatizing compound, it is reasonable to expect
the derivatized oligosaccharides (e.g., glycoprotein released
oligosaccharides, proteoglycans) would retain their sensitivity to
other exoglycosidases (e.g., neuraminidase, mannosidase,
N-acetylgalctosaminidase) which act at site quite removed from the
derivatizing compound. Partial digestion using .beta.-galactosidase
resulted resulted in three bands corresponding to trisaccharide-AGA
starting material a disaccharide-ACA conjugate and a
monosaccharide-AGA conjugate. This result gives the sequence of the
trisaccharide starting material and suggests that it is possible to
use their approach to sequence more complex oligosaccharides. The
strategy involves: 1) release of oligosaccharides from
glycoproteins using an endoglycosidase, such as N-glycanase; 2)
conjugation of the released oligosaccharides to AGA; 3)
fractionation and purification of each oligosaccharide-AGA
conjugate; 4) sequential treatment of each purified
oligosaccharide-AGA conjugate with specific exoglycosidases; and 5)
analysis by analytical PAGE and reading of the sequence from the
observed banding pattern.
Example 2
Sequencing Lacto-N-tetraose
AGA was coupled to the reducing end of lacto-N-tetraose (Gal(.beta.
1.fwdarw.3)GlcNAc(.beta. 1.fwdarw.3)Gal(.beta. 1.fwdarw.4)Glc)
(purchased from Sigma Chemical) using the conditions described in
Example 1. The lacto-N-tetraose derivative thus prepared was
purified by gel electrophoresis and recovered by electrotransfer,
as described in Example 1. Freeze-dried, desalted AGA derivative of
lacto-N-tetraose (0.5 .mu.g) was analyzed by gel electrophoresis:
1) directly with no enzymatic treatment; 2) following 12 h
treatment with 100 munit .beta.-galactosidase (A. Niger); 3)
following treatment with .beta.-galactosidase (as in 2), thermal
inactivation of .beta.-galactosidase (100.degree. C., 2 min), and
12 h treatment with 10 munit N-acetylglucosaminidase (jack bean);
and 4) following treatment with .beta.-galactosidase, thermal
inactivation, treatment with N-acetyl glucosaminidase (as in 3),
thermal inactivation of N-acetylglucosaminidase (100.degree. C., 2
min), and treatment with .beta.-galactosidase (A. niger).
Individual lanes showed bands corresponding to labeled
tetrasaccharide, labeled trisaccharide, labeled disaccharide and
labeled monosaccharide. From the known specificity of the enzymes,
the sequence for lacto-N-tetraose could be established.
Example 3
Analysis of Oligosaccharides Having a Charged Label Using Capillary
Zone Electrophoresis
A Capillary Electrophoresis System I (Dionex), equipped with an
unfilled capillary (CZE) (65 cm, 75 .mu.m ID, 375 .mu.m OD) and
using fluorescence detection was used to analyze the
monosaccharide-AGA derivative, the disaccharide-AGA derivative and
the trisaccharide-AGA derivative described in Example 1. Gravity
injection was used to apply 10 nl of sample (10 .mu.g/.mu.l) a
voltage of 17 kV with 17 .mu.Amp current was applied using negative
polarity. The buffer system used was 20 mM citric acid, pH 2.0.
Peaks corresponding to each of the AGA derivatives were detected.
This experiment demonstrates that it is also possible to use
capillary electrophoresis to sequence oligosaccharides. Capillary
electrophoresis has the advantage of permitting easy automation of
this sequencing approach.
Equivalents
Those skilled in the art will know, or be able to ascertain using
no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. These and
other equivalents are intended to be encompassed by the following
claims.
* * * * *